Hendra virus and Nipah virus are highly pathogenic zoonotic paramyxoviruses. Recently emerged from flying foxes, these viruses cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore, Bangladesh and India. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. Mortality rates are high in both viruses yet there is no vaccine or therapeutic approved for human or livestock use.
Since the virus genome encodes only 6 genes, host cell factors are critical for successful replication of the virus and are involved in all stages of the HeV life cycle. The application of RNAi seeks to inhibit specific gene products. In mammalian cells, silencing of cellular genes can be achieved by transfection of synthesised small-interfering RNA molecules. In brief, small interfering RNAs (siRNAs) are introduced into cells and loaded into the RNA induced silencing complex (RISC). Single strand RNA binds to RISC and dictates the binding to a target mRNA. Perfect complementarity results in site-specific cleavage of the target mRNA, effectively silencing the gene product. Assayed data can be directly translated towards the identification of potential antiviral drug targets.
We will attempt to silence each individual gene in the human cell to determine those genes involved with Hendra virus replication. Facilities at the Victorian Centre for Functional Genomics at the Peter MacCallum Cancer Centre include robotics for the transfection and cell seeding in 384 well microplates and assessment of cell viability. At CSIRO AAHL, the plates will be infected and assayed for Hendra viral growth. Validation will include testing of the four individual siRNA duplexes comprising each hit pool.